EP3084447A1 - Identification de dimension de sous-forme lp(a) à l'aide d'électrophorèse d'immuno-fixation de gel zonal - Google Patents

Identification de dimension de sous-forme lp(a) à l'aide d'électrophorèse d'immuno-fixation de gel zonal

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Publication number
EP3084447A1
EP3084447A1 EP14828097.7A EP14828097A EP3084447A1 EP 3084447 A1 EP3084447 A1 EP 3084447A1 EP 14828097 A EP14828097 A EP 14828097A EP 3084447 A1 EP3084447 A1 EP 3084447A1
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European Patent Office
Prior art keywords
subforms
individual
sample
apo
gel
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP14828097.7A
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German (de)
English (en)
Inventor
Philip Guadagno
Erin Grace SUMMERS
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Helena Laboratories Corp
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True Health Diagnostics LLC
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Publication of EP3084447A1 publication Critical patent/EP3084447A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Electrophoresis is a technique used to separate charged species on the basis of size, electric charge, and other physical properties.
  • the charged species migrate through a conductive electrophoretic medium, which may be (but is not required to be) a gel, under the influence of an electric field.
  • Activated electrodes located at either end of the electrophoretic medium provide the driving force for the migration.
  • the properties of the molecules, including their charge and mass, determine how rapidly the electric field causes them to migrate through the electrophoretic medium.
  • the frictional force of the electrophoretic medium also aids in separating the molecules by size.
  • the molecules may be stained. After staining, the separated macromolecules can be seen in a series of bands spread from one end of the electrophoretic medium to the other. If these bands are sufficiently distinct, the molecules in these zones can be examined and studied separately by fixing macromolecules and washing the electrophoretic medium to remove non-fixed components and remaining buffer solution.
  • apolipoproteins and/or lipoprotein particles including, but not limited to, cardiovascular disease, Alzheimer's disease, hyperlipidemia, abetalipoproteinemia, hypothyroidism, liver disease, diabetes mellitus, and renal problems. Accurate predictors of the risk of an individual of developing various diseases related to lipoprotein particles are needed for research, diagnostic, and therapeutic purposes.
  • Apo(a) is one such protein that partly comprises the Lp(a) particle.
  • the present invention is directed to overcoming these and other deficiencies in the art.
  • a method for determining the composition of individual Lp(a) subforms in a test sample involves: (a) providing a test sample comprising Lp(a) subforms obtained from a subject; (b) separating the Lp(a) subforms in the test sample along an electrophoretic gel; (c) measuring the migration velocity of the individual Lp(a) subforms along the electrophoretic gel; (d) comparing, based on the measuring, the migration velocity of the individual Lp(a) subforms to a reference value; and (e) determining, based on the comparing, the molar mass of the individual Lp(a) subforms.
  • a method for predicting cardiovascular health involves obtaining a sample from a patient; measuring the size distribution of Lp(a) subforms in the sample; characterizing the patient's risk of cardiovascular disease based on Lp(a) subform sizes and/or distribution.
  • a method for predicting cardiovascular health involves obtaining a sample comprising Lp(a) subforms from a patient; separating the Lp(a) subforms in the test sample along an electrophoretic gel; measuring particle number of individual Lp(a) subforms in the sample; and determining a cardiovascular risk value for the subject based on the measured particle number of the Lp(a) subforms.
  • Lipo-IFE lipoprotein immuno-fixation electrophoresis
  • a simple rapid single zonal gel IFE method is described that is suitable for population screening to provide both Lp(a) particle number and apo(a) subform size.
  • Methods described herein have the advantage of not only a short time to analyze a sample (approximately 90 minutes), but they are also cost-effective. Such a method is a significant improvement on existing technology.
  • FIGS. 1 A and IB show variable Lp(a)-P migration rates via the results of a Lipo-IFE protocol, as described herein.
  • FIG. 1 A provides a basic example gel for evaluating differences in Lp(a) migration.
  • FIG. 1A labels electrophoretic banding on the example gel corresponding to LDL, vLDL, median Lp(a), slower cathodic Lp(a), and faster anodic Lp(a).
  • FIG. IB the results of 5 samples run according to the protocols in this application are shown. Samples all contain LDL and VLDL, and all but the negative control further includes Lp(a) particles.
  • the reference (known) Lp(a) content comprises an apo(a) moiety of 600-650 kD.
  • an "anodal" sample in lane 2 contains Lp(a) with an apo(a) moiety of greater than 650 kD
  • the "mid” sample in lane 3 contains apo(a) moieties of the same mass as the reference, 600-650kD
  • the "cathodal” sample contains apo(a) moieties of less than about 600 kD.
  • FIGS. 1A and IB illustrate the principle of differential detection and quantification of Lp(a) subforms.
  • FIG. 2 shows examples of samples with differential Lp(a)-P migration bias on zonal gels, separated via the Lipo-IFE protocol described herein.
  • a series of samples have been run on a gel in parallel on the Lipo-IFE system.
  • the cathode and anode ends of the gel are labeled and solid lines represent the position of Lp(a) particles after separation where apo(a) mass is about 600-650 kD, that distinguishes large (anodal) from small (cathodal) particles.
  • Four samples have been highlighted (sample numbers 10, 73, 24, and 44).
  • samples 10 and 73 show distinct Lp(a) subform size difference due to the migration rates of smaller subforms (samples 10 and 73, with dashed outline and positioned toward the cathodal end of the substrate) and larger subforms (samples 24 and 44, with a solid outline and positioned toward the anodal end of the substrate).
  • DBL indicates the presence of two apo(a) isoforms in a lane.
  • FIG. 3 compares the zonal gel (Lipo-IFE protocol, inset) shown in
  • FIG. 2 to the same samples in a western blot.
  • samples 24, 73, 44, and 10 are further analyzed in a western blot analysis after apo(a) removal from the Lp(a) particles.
  • Western Blot analysis was carried out using standard protocols with Apolipoprotein(a) Isoform Analysis (AAISO), using the Novex
  • Sample 24 which is an anodal (or larger) Lp(a) particle, exhibits a larger separated apo(a) in the western blot, appearing at around 700kD and greater than 700 kD.
  • Sample 73 which is a cathodal, (or a smaller particle in the zonal gel) corresponds to a smaller apo(a) moiety around 600 kD in the western blot.
  • Samples 44 and 10 repeat the pattern with an anodal Lp(a) similarly having larger apo(a) bands at -700 kD and a cathodal Lp(a) having a smaller apo(a) band at less than 450 kD on the western blot.
  • DBL indicates the presence of two apo(a) isoforms in a lane.
  • FIGS. 4A and 4B show more examples of the Lp(a)-P migration differentials in a high-throughput run (see Example 1 and Guadagno et al., "Validation of a Lipoprotein(a) Particle Concentration Assay by Quantitative Lipoprotein Immunofixation Electrophoresis,” Clin Chim Acta 439:219-224 (2014), incorporated herein by reference in its entirety).
  • High-throughput involves the parallel analysis of a multiplicity of samples on automated instrumentation, in this case the Lipo-IFE (Helena Laboratories). In this experiment, 131 samples were tested, including 2 mouse variants.
  • Samples were chosen based upon their electrophoretic migration velocity (to confirm migration velocity and apo(a) size relationship) and Lp(a)-P to mass indices.
  • zonal gels run in parallel show variation among patient samples in a high- throughput experiment.
  • Anodal particles of more than about 650 kD and cathodal particles of less than about 600 kD are identified.
  • Anodal samples include sample numbers 1 147, 1200, 0481 , 1621, 2420, 2495, and 2618.
  • Cathodal samples include sample numbers 1 101 and 261 1.
  • FIG. 5 shows comparison of apo(a) content and apoB content in a series of samples on a zonal gel.
  • five samples were probed by anti- apoB sera and anti-apoA sera, showing doublet banding, a product of some subjects having two sizes of apo(a) moieties on their Lp(a) particles.
  • Lp(a) doublet banding is seen on samples 1 , 3, 4 & 5; these samples contain both apoB & apo(a) probe response without non-specific protein residue in saline lane. Doublet banding is not artifactual and confirms the sensitivity and resolving power of the present methods.
  • Lp(a) doublet banding can be equal, primarily cathodal, or anodal with differential resolution.
  • Sample 2 has single Lp(a), but contains both apoB and apo(a) response, cathodal to LDL and without nonspecific protein residue in the saline lane.
  • the frequency of doublet banding is ⁇ 1 %. Saline probe is used to establish presence of artifactual banding.
  • FIGS. 6A-6R present data comparing Lp(a)-P zonal migration velocities (inset, from FIGS. 2, 4A, and 4B) associated with increasing MW of apo(a), measured by western blot.
  • FIGS. 6A-6R more than 100 samples with Lp(a) zonal migration biases were compared to apo(a) isoform size analysis by western blot analysis, as described above.
  • the results from 19 experiments reflecting the same setup and analysis as shown in FIG. 3 are presented.
  • the results show consistent agreement of the new zonal gel method for analyzing Lp(a)-P subform size with the more intensive analysis of separated apo(a) moieties from the same particles.
  • "DBL" indicates the presence of two apo(a) isoforms in a lane.
  • FIG. 7 shows fluorescent imaging for labeled anti-apoB antibodies bound to Lp(a), LDL, and VLDL particles.
  • the image is a 3Blot/TBS-Image (wet) (as also described in Guadagno et al., "Validation of a Lipoprotein(a) Particle Concentration Assay by Quantitative Lipoprotein Immunofixation
  • Electrophoresis Clin Chim Acta 439:219-224 (2014), incorporated herein by reference in its entirety).
  • the gel blocks were removed and a rigid antisera template was placed on the gel.
  • the antibody was diluted 1 :4 with normal saline and administered through the template onto the gel for 2 min. Excess antibody was removed by blotting and pressing. Residual matrix antibody was removed by rehydration of the gel in a tris-buffered saline bath for 1 min. These steps were performed three times.
  • the gel was subsequently dried at 56° for 8 min, then stained with Acid Violet and scanned.
  • the top row shows sample 3022 at the dilution levels of 5: 1 (Al), 7.5: 1 (A2), and 10: 1 (A3) Alexa Fluor® fluorescent dye:polyclonal antibody, in respective columns.
  • the same experiment is shown for sample 3052 in the bottom row. There are no observable differences in optical properties between the dilution ratios.
  • FIGS. 8A-8E show results of an initial comparison of acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods (wherein anti-apoB antibodies are labeled with a fluorescein derivative such as Alexa Fluor® 488 or similar and incubated with the lipoproteins before washing and analyzed), for Lp(a)-P distinction from LDL-P in a single sample.
  • FIG. 8A shows a native zonal gel separation of sample numbers 3022 and 3052, in respective columns containing duplicate runs, labeled by acid violet staining. There are 4 total sample runs, where the LDL is evident as a strong band and Lp(a) as a weak band below it.
  • FIG. 8B shows optical imaging with fluorescence of the same samples, where 3022 is on the top row and 3052 is on the bottom row. The columns correspond to different fluorescent dye:anti- apoB ratios in reagents Al , A2, and A3, previously described.
  • FIG. 8C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low-density lipoprotein) of the total detected sample.
  • FIG. 8D is the optical density reading of the acid violet staining of sample 3022 in profile
  • FIG. 8E is the optical density reading of the fluorescent conjugate of sample 3022 in profile (in the reverse direction of the A/V profile).
  • FIGS. 9A-9E show results of an initial comparison of acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 9A shows a native zonal gel separation of samples 3034 and 3051, in respective columns containing duplicate runs, labeled by acid violet staining. There are 4 total sample runs, where the LDL is evident as a strong band and Lp(a) as a weak band below it.
  • FIG. 9B shows optical imaging with fluorescence of the same samples, where 3034 is on the top row and 3051 is on the bottom row.
  • FIG. 9C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 9D is the optical density reading of the acid violet staining of sample 3051 in profile
  • FIG. 9E is the optical density reading of the fluorescent conjugate of sample 3051 in profile (in the reverse direction of the A/V profile).
  • FIGS. l OA- 10E show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A/V) and apoB*-fluorescently-tagged antibody labeling methods for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 10A shows a native zonal gel separation of sample 0816, in the left column, labeled by acid violet staining. On the left, anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and VLDL.
  • FIG. 10B shows optical imaging with fluorescence of the same sample where labeled. The columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al, A2, and A3, previously described.
  • FIG. IOC presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 10D is the optical density reading of the acid violet staining of sample 0816 in profile
  • FIG. 10E is the optical density reading of the fluorescent conjugate of sample 0816 in profile (in the reverse direction of the A/V pro file).
  • FIGS. 1 lA-1 IE show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A/V) and apoB* fluorescently-tagged labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 1 1 A shows a native zonal gel separation of sample 2377, in the left column, labeled by acid violet staining. On the left, anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and VLDL.
  • FIG. 1 I B shows optical imaging with fluorescence of the same sample where labeled. The columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al , A2, and A3, previously described.
  • FIG. 1 1C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 1 D is the optical density reading of the acid violet staining of sample 2377 in profile
  • FIG. 1 I E is the optical density reading of the fluorescent conjugate of sample 2377 in profile (shown in reverse sequence from A/V).
  • FIG. 12A-12E show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 12A shows a native zonal gel separation of sample 3389, in the left column, labeled by acid violet staining.
  • anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and VLDL.
  • anti-apo(a) antibodies are used, which only label the Lp(a) particles.
  • FIG. 12B shows optical imaging with fluorescence of the same sample where labeled.
  • the columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al, A2, and A3, previously described.
  • FIG. 12C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 12D is the optical density reading of the acid violet staining of sample 3309 in profile
  • FIG. 12E is the optical density reading of the fluorescent conjugate of sample 3389 in profile (shown in reverse sequence from A/V).
  • FIGS. 13A-13C shows a summary of figures 10-12, comparing doublet-containing samples 0816, 3389, and 2377 to each other on adjacent gels.
  • FIG. 13A shows the zonal gels labeled with acid violet stain for each sample, with apoB labeled in the first column and apo(a) labeled in the second column.
  • FIG. 13B shows the fluorescence detection for each sample, with the samples clearly labeled.
  • FIG. 13C presents the table summarizing each sample and its relative proportion of anodal (large) Lp(a) subform and cathodal (small) Lp(a) subform along with the LDL and VLDL portions of the sample. The methods show good agreement in calculated ratios of each lipoprotein type level independent of subform type and/or lipoprotein type or level.
  • a method for determining the composition of individual Lp(a) subforms in a test sample involves: (a) providing a test sample comprising Lp(a) subforms obtained from a subject; (b) separating the Lp(a) subforms in the test sample along an electrophoretic gel; (c) measuring the migration velocity of the individual Lp(a) subforms along the electrophoretic gel; (d) comparing, based on the measuring, the migration velocity of the individual Lp(a) subforms to a reference value; and (e) determining, based on the comparing, the molar mass of the individual Lp(a) subforms.
  • apo(a) is one such protein that partly comprises the Lp(a) particle.
  • Apo(a) may comprise a range of sizes due to the repeats of a particular sequence of amino acids in the protein, a region described as having kringle repeats. See Lackner et al., "Molecular Basis of Apolipoprotein(a) Subform Size Heterogeneity as Revealed by Pulsed-Field Gel Electrophoresis," J Clin Invest 87:2153-61 (1991); Lackner et al., "Molecular Definition of The Extreme Size Polymorphism in Apolipoprotein(a),” Hum Mol Genet 2:933-940 (1993), each of which is hereby incorporated by reference in its entirety.
  • the number of kringle repeats in apo(a) may range from 10 to greater than 50 repeats. See id.
  • the various sizes of apo(a) due to kringle repeats are called apo(a) subforms or isoforms.
  • Lp(a) is known to be a risk factor for cardiovascular disease and an increase in the number of kringle repeats is inversely correlated with Lp(a) concentration in most, but not all cases.
  • the size of Lp(a) particles in the blood may have significant importance for cardiovascular health. See Rifai et al., "Apolipoprotein(a) Size and Lipoprotein(a) Concentration and Future Risk of Angina Pectoris with Evidence of Severe Coronary
  • cardiovascular risk may involve assigning the subject to one of a low, moderate, or high cardiovascular risk category.
  • biochemical markers for determining risk there are well established recommendations for cut-off values for biochemical markers for determining risk ⁇ see Rifai et al., "Apolipoprotein(a) Size and Lipoprotein(a) Concentration and Future Risk of Angina Pectoris with Evidence of Severe Coronary Atherosclerosis in Men: The Physicians' Health Study," Clin. Chem. 58(8): 1364-1371 (2004); Erqou et al., "Apolipoprotein(a) Isoforms and the Risk of Vascular Disease," J. Am. Coll.
  • a risk assessment based solely on Lp(a) particle number may be modified by the predominance of large or small isoforms.
  • the isoform size can be used as a weighting factor among a plurality of additional markers.
  • the isoform size may be evaluated
  • predominantly small isoforms may be assigned to a high-risk category, while predominantly large isoforms (e.g., more than about 640kD) may be assigned to low-risk category.
  • the isoform cutoff between small and large distinctions may vary in the range of 600kD to 700kD.
  • the difference between small and large isoform size may be characterized at, for example, about 600kD, 605kD, 610kD, 615kD, 620kD, 625kD, 630kD, 635kD, 640kD, 645kD, 650kD, 655kD, 660kD, 665kD, 670kD, 675kD, 680kD, 685kD, 690kD, 695kD, or 700kD.
  • high-, moderate-, and low-risk particle sizes may be separated into categories of about ⁇ 600kD, about 600-640kD, and about >640kD, respectively.
  • high-, moderate-, and low-risk particle sizes may be separated into categories of about ⁇ 600kD, about 600-700kD, and about >700kD, respectively.
  • a patient's risk may also be characterized by low risk if they have two large-isoform versions of the apo(a), by moderate risk if they have one large and one small isoform, and high risk if they have two small isoforms.
  • a patient may be characterized as low-, moderate-, or high-risk by taking an average weight of the isoforms present in their sample and categorizing the average as low-, moderate-, or high-risk by their location among a population values split into tertiles.
  • Another method for stratification may involve the estimation of kringle IV repeats and stratifying risk according to tertiles of the number of kringle IV repeats on an apo(a).
  • the high-risk category may include apo(a) with less than 19 kringle repeats, moderate-risk with 19-29 kringle repeats and low-risk greater than 30 kringle repeats.
  • the risk categories need not be symmetric. In many cases, only the top quintile is considered high-risk, which would involve fewer than 19 kringle repeats, and the moderate and low-risk categories are split among the remaining population distribution.
  • the categories may be determined by a longitudinal study comparing outcomes versus apo(a) sizes among patients in a study.
  • Apolipoprotein a (“apo(a)") is highly heritable and mainly controlled by the apo(a) gene [LPA] located on chromosome 6q26-27. Apo(a) is co-dominantly expressed and therefore both should be present and detectable for a patient with genes for two different isoforms. Doublets in the AAISO system and method presented herein confirm its sensitivity and resolving power through the precise control of migration velocity. The method permits population studies to further validate the apo(a) diversity among patients.
  • a method to determine the molecular weight ("MW") of apo(a) on intact/native Lp(a)-Particles i.e., non- denatured.
  • the term molecular weight may refer to molar mass.
  • Intact or native Lp(a)-Particles include those that have not been subjected to denaturing treatment such as treatment with sodium dodecyl sulfate (“SDS”) delipidation, reduction or removal of the apolipoprotein from the intact particle.
  • SDS sodium dodecyl sulfate
  • an algorithm may be established for CVD risk relative to Lp(a) particle number mitigated by subform size.
  • V the rate (velocity) of migration
  • E the strength of the electrical field
  • Z the charge on the molecule
  • F the frictional force on the molecule.
  • cations (positively charged) in solution migrate toward the cathode of gel electrophoresis (negatively charged) whereas anions (negatively charged) migrate toward the anode of gel electrophoresis (positively charged) when an electrical field is applied.
  • the migration velocity is proportional to the ratio between the charges of the protein and its mass. The higher charge per unit of mass, the faster the migration.
  • proteins are denatured by adding a detergent such as SDS, to separate them exclusively according to molecular weight (Shapiro et al., "Molecular Weight Estimation of Polypeptide Chains by Electrophoresis in SDS-Polyacrylamide Gels," Biochem. Biophys. Res. Commun. 28: 815-820 (1967), which is hereby incorporated by reference in its entirety).
  • SDS is a mild reducing agent which maintains the polypeptides in a charged denatured state once the protein has been exposed to strong reducing agents to reduce the disulfide bonds to sulfhydryls.
  • Lp(a)-P is a lipid particle with a single apoB, (MW ⁇ 540kD) and a single apo(a). Subforms of apo(a) however can vary from 3 OOkD to 900kD.
  • the overall charge on the Lp(a)-P is the sum of the charges from both apoB and the particular polymorphic isoform of apo(a).
  • apoB has a constant MW
  • any variation in charge for the Lp(a)-P will be a function of apo(a) size.
  • Migration velocity (V is proportional to Z) will be a function of the differential charge from the apo(a) subform, which is directly proportional to apo(a) size (i.e., apo(a) MW).
  • lipoprotein particle refers to a particle that contains both protein and lipid. Examples of lipoprotein particles are described in more detail below.
  • lipoprotein particle number refers to the molar concentration (nmol/L) of lipoprotein particles present in the bodily fluid. Particle number may be measured as molar concentration in nmol/L.
  • apolipoprotein refers to a protein that combines with lipids to form a lipoprotein particle. Examples of apolipoprotein types are described in more detail below. The unique nature of the apolipoprotein is their stoichiometric relationship to lipoprotein particles, providing an estimate of the lipoprotein particle number, which is described in more detail below.
  • Suitable biological samples or biosamples according to the invention include human biological matrices, urine, cerebrospinal fluid, whole blood, plasma, serum, and human lipoprotein fractions.
  • the sample may be fresh blood or stored blood or blood fractions.
  • the sample may be a blood sample expressly obtained for the assays of this invention or a blood sample obtained for another purpose which can be subsampled for use in accordance with the methods according to the invention.
  • the biological sample may be whole blood. Whole blood may be obtained from the subject using standard clinical procedures.
  • the biological sample may also be plasma. Plasma may be obtained from whole blood samples by centrifugation of anti-coagulated blood.
  • the biological sample may also be serum.
  • the sample may be pretreated as necessary by dilution in an appropriate buffer solution, concentrated if desired, or fractionated by any number of methods including but not limited to
  • Methods as described herein may be used with any suitable gel electrophoresis system and/or method.
  • Suitable gel electrophoresis systems and methods include, for example, those described in WO 2013/181267 and U.S. Patent Application Publication No. 2012/0052594, each of which is hereby incorporated by reference in its entirety.
  • Apparatuses for the detection of Lp(a)-P that may be used in accordance with methods as described herein include those of U.S. Patent Application Publication No. 2012/0052594, which is hereby incorporated by reference in its entirety.
  • immunofixation methods such as described in U.S.
  • a biological sample e.g., serum
  • Anti- sera containing labeled antibodies e.g., anti-ApoB antibodies
  • the antibodies attach to their antigen targets, and the targets can be identified through some means of detecting the label.
  • methods described herein may also employ unlabeled antibodies that are detected by contacting the gel with a protein dye (e.g., acid violet or the like). Accordingly, the methods described herein may involve contacting the electrophoretic gel with a protein dye that dyes apoB on the individual Lp(a) particles and subsequently detecting the dyed Lp(a) subforms. Dye binding is associated with dye bound to anti-apoB to apoB. This represents the primary signal. Dye bound to apo(a) is secondary signal noise relative to the signal produced by the PAb-apoB-A/V.
  • the protein dye may be acid violet.
  • the protein dye may also be a Coomassie dye, a derivative or improvement thereof such as Bio-Safe® (Bio-Rad, Hercules, Calif.) or SimplyBlue® (Invitrogen, Carlsbad, Calif.) or Imperial Protein Stain (Thermo Fisher Scientific, Rockford, 111.), a silver-based stain, a zinc-based stain, a fluorescent dye, or there may be a functional group specific stain such as glycoprotein stain, phosphoprotein stain, His-tag stain, or any other type or combination of stains.
  • Such methods are well known by those skilled in the art, as described in, for example, H.J. CONN'S BIOLOGICAL STAINS (R.D.
  • methods described herein may involve fixing the separated individual Lp(a) subforms within the gel prior to detection.
  • Fixing the separated individual Lp(a) subforms may be carried out by contacting the gel with a suitable antibody (or anti-sera containing antibodies), as is well-known.
  • the antibody may be an anti-ApoB antibody.
  • fixation may be followed by contacting the gel with a protein stain (such as acid violet, as described herein) and detection of the stained Lp(a)-P subforms using a densitometer.
  • the gel electrophoresis may be one-dimensional or two- dimensional. Isoelectric focusing may also be performed. Electrophoretic gel substrates suitable for use with the invention are known to those of skill in the art. For instance, suitable gel substrates include, but are not limited to, agarose or poiyacrylamide or blends of the two. As discussed above, SDS-PAGE
  • (poiyacrylamide) gels separate proteins based on their size because the SDS coats the proteins with a negative charge. Separation of proteins on the agarose gel is by charge. Accordingly, as also noted above, embodiments described herein may use zonal gel electrophoresis. Zonal gel electrophoresis, wherein non-denatured proteins are separated by charge offers the benefit of a simple high-resolution protocol.
  • Electrophoretic gels of varying sizes may contain various numbers of lanes and rows (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.).
  • the biological sample from a single individual or subject may be probed to identify multiple components and/or serum from multiple individuals may be tested.
  • the protocols for conducting electrophoresis on different sizes of gels will be similar except that modifications may be made to optimize separation on that size of gel.
  • Methods according to aspects described herein may also include characterizing, based on the determining of the molar mass of the individual Lp(a) subforms, at least two Lp(a) subforms of different molar mass.
  • Methods according to aspects described herein may also include measuring the total particle number of the Lp(a) present in the sample, the individual Lp(a) subforms, or both.
  • electrophoresis measures relative concentrations, i.e., percentage fractions are calculated as the area under curves from detected bands that have been translated into signals to produce
  • a gel may be stained with a protein dye (e.g., Acid Violet) and passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands.
  • a densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a stained gel. Absorbance can be measured with densitometry and fluorescence can be measured with photon- counting imaging protocols such as provided by BioRad Chemidoc® instruments. Using the optical density measurements, the densitometer represents the detected bands of stain as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display.
  • microprocessor evaluates the area under each peak and reports each as a percent of the total sample. For example, if the electrophoresis is being used for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration.
  • the particle number of the individual Lp(a) subforms may also be determined by measuring the apoB concentration of the particular subform separated along the electrophoretic gel. The particle number may be quantified by comparison with a separate analysis that characterizes the total lipid particle or class of lipid particle concentration in the sample. Such separate analysis may be ultracentrifugation (UC), NMR, or any other analysis method that can characterize a concentration or total particle number for particles in the sample.
  • UC ultracentrifugation
  • NMR nuclear magnetic resonance
  • Densitometer software may also automatically calculate and print the relative percent and the mg/dL for each band along an electrophoretic gel when the specimen total Apo-B is entered as (% of Fraction) x (total Apo-B).
  • the conversion factor (mg/dL) / 0.054 is calculated from:
  • Conducting electrophoresis may involve carrying out the step of depositing a sample in a receiving well of an electrophoretic gel as part of a method for assessing the level of and/or molecular mass of the Lp(a) subforms present in a bodily fluid, as described in U.S. Patent Application Publication No. 2012/0052594, which is hereby incorporated by reference in its entirety.
  • the exemplary method involves separating lipoprotein particles present in a bodily fluid sample by gel electrophoresis on a gel electrophoresis substrate, exposing the substrate to an antibody to detect an immunologically active agent associated with lipoprotein particles or components of lipoprotein particles, exposing the substrate to a reagent for detection of the presence of proteins or lipids, and determining the level and/or molecular mass of the Lp(a) subforms.
  • Methods described herein may also be carried out in conjunction with in-situ calibration (as described in U.S. Patent Application Publication No. 2013/0319864, which is hereby incorporated by reference in its entirety) and involve combining a volume of a test sample with a volume or quantity of a calibrating sample to form a final volume, in which the volume or quantity of the calibrating sample includes a known concentration of a calibrator and the final volume includes a known ratio of test sample to calibrating sample.
  • a known mass of dry calibrator may be mixed into the test sample to provide a known concentration of calibrator and no volume change.
  • the method also includes depositing a loading fraction in a receiving well, electrophoretic gel, in which the loading fraction is a fraction of the final volume and separating the loading fraction along a common separation lane of the electrophoretic gel such that components of the test sample and the calibrator are separated from one another along the common separation lane.
  • the method also includes detecting the calibrator and separated components of test sample within the common separation lane and measuring the level of the calibrator and separated components of the test sample based on the detecting, thereby performing electrophoresis with in-situ calibration.
  • the reference value as described herein may be a control Lp(a) subform separated along the electrophoretic gel.
  • the reference value may also be a predetermined value used for comparison.
  • the method can distinguish Lp(a) particles with apo(a) proteins of molecular weights, for example, greater than 700kD, less than 600kD and between 600 and 700kD.
  • the molar mass of apo(a) protein of the individual Lp(a) subforms is greater than 600kD.
  • the molar mass of apo(a) protein of the individual Lp(a) subforms is determined to be greater than 700kD.
  • the molar mass of apo(a) protein of the individual Lp(a) subforms is between 600 and 700kD.
  • the molar mass of apo(a) protein of the individual Lp(a) subforms is less than 600kD.
  • Determining the molar mass of the individual Lp(a) subforms may involve assigning the individual Lp(a) subforms to one of a low, mid, or high molar mass category. For instance, the individual Lp(a) subforms having a molar mass less than about 600kD may be assigned to the low molar mass category; individual Lp(a) subforms having a molar mass of between about 600kD and 700kD may be assigned to the mid molar mass category; and individual Lp(a) subforms having a molar mass of greater than about 700kD are assigned to a high molar mass category.
  • the individual Lp(a) subforms of the test sample may each be bound to a signal-producing molecule capable of producing or causing production of a detectable signal.
  • the method also involves contacting the separated individual Lp(a) subforms of the test sample bound to the signal-producing molecule with a reagent capable of interacting with the signal-producing molecule, where the signal-producing molecule produces the detectable signal upon contact with the reagent and where said method further comprises detecting the detectable signal.
  • the individual Lp(a) subforms of the test sample are bound to signal-producing molecules that are distinguishable from one another.
  • Suitable systems and methods involving signal-producing molecules that are distinguishable from one another include those for use in in situ detection of lipid particles within an electrophoretic matrix, as describe in U.S. Patent Application Publication No. 2014/0243431, which is hereby incorporated by reference in its entirety.
  • Such a system includes a gel substrate to receive a biological sample and at least two lipoprotein-binding complexes.
  • Each complex includes an antibody that binds a lipoprotein particle or a portion thereof, where the antibody is bound to a signal-producing molecule capable of producing or causing production of a detectable signal.
  • Each detectable signal of the at least two lipoprotein-binding complexes is distinguishable from the other detectable signal.
  • the system also includes a device for detecting the detectable signal, where the detecting indicates the level of the specific Apolipoproteins and/or lipoprotein particles in the biological sample.
  • antibody is meant to include intact immunoglobulins derived from natural sources or from recombinant sources, as well as immunoreactive portions (i.e. antigen binding portions) of intact immunoglobulins.
  • the antibodies of the invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies, antibody fragments (e.g., Fv, Fab and F(ab)2), as well as single chain antibodies (scFv), chimeric antibodies and humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999); Houston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc Natl Acad Sci USA 85:5879-5883 (1988); Bird et al, "Single-Chain Antigen-Binding Proteins," Science 242:423-426 (1988), which are hereby incorporated by reference in their entirety).
  • polyclonal antibodies may be produced by injecting a suitable animal host, such as a rabbit, with the lipoprotein of interest and an adjuvant. Approximately 0.02 milliliters may be injected, with reinjection occurring every 21 days until peak antibody titer is achieved. Antibody titer may be tested by, for example, an ear bleed. Antibodies to Apo B-100 or other apolipoprotein may be produced in this manner. Alternatively, antibodies to Apo B-100 or other apolipoprotein may be purchased commercially.
  • Antibodies can be generated with high levels of specificity, sufficient to distinguish different portions of the same proteins, such as different kringles on apo(a), in particular repeating kringle IV and any other kringle on apo(a). As Lp(a) subforms are distinguished by the number of kringle repeats, characterizing kringle content can facilitate identification of Lp(a) size with great detail. Such antibodies would be labeled with, for example, different color fluorescent probes (as described above) and the apolipoprotein type can be distinguished with extreme detail. As described below, absolute levels and ratios of detailed measurements can be reported and converted into a risk factor. For example the ratio of small to large Lp(a) can be reported with a specific cutoffs for high-, medium-, and low-risk ranges.
  • the invention encompasses binding portions of such antibodies.
  • binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), single variable VH and V L domains, and the bivalent F(ab') 2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.
  • Suitable signal-producing molecules that are capable of producing or causing production of a detectable signal will be known to those of skill in the art.
  • the detectable signal includes any signal suitable for detection and/or measurement by radiometric, colorimetric, fluorometric, size-separation, or precipitation means, or other means known in the art.
  • Examples of signal-producing molecules that are capable of producing or causing production of a detectable signal include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.
  • the signal-producing molecules may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the invention. Further examples include, but not limited to, various enzymes.
  • Examples of enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin.
  • Examples of fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • Examples of luminescent material include, but are not limited to, luminol.
  • bioluminescent materials include, but not limited to, luciferase, luciferin, and aequorin.
  • radioactive material include, but are not limited to, bismuth (213Bi), carbon (14C), chromium (51Cr), (153Gd, 159Gd)5 gallium (68Ga, 67Ga), germanium (68Ge), holmium (166Ho), indium (1 151n, 1 13In, 1 121 ⁇ , 1 1 1 In), iodine (131 1 , 1251 , 1231, 1211), lanthanium (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (103Pd), phosphorous (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthemium (97Ru), samarium (153
  • Detection of an antibody-signal producing molecule complex in accordance with the invention may also be achieved by addition of a reagent capable of interacting with the signal-producing molecule, where the signal- producing molecule produces a detectable signal upon contact with the reagent. For example, light is emitted when luciferase acts on the appropriate luciferin substrate.
  • a secondary antibody that is coupled to a detectable signal or moiety such as for example, an enzyme (e.g., luciferase), fluorophore, or chromophore may also be used.
  • an enzyme e.g., luciferase
  • fluorophore e.g., fluorophore
  • chromophore e.g., chromophore
  • each detectable signal of the at least two lipoprotein-binding complexes may be distinguishable from the other detectable signal. This permits cocktailing at least two lipoprotein-binding complexes where each of the complexes detects a different Lp(a) subform or a portion thereof, each complex also producing or capable of producing a different detectable signal.
  • a first lipoprotein-binding complex may include fluorescein
  • a second non-kringle IV-binding complex may include rhodamine-labeled antibody which binds a second portion of apo(a).
  • the first and second complexes may be mixed or cocktailed together. This permits probing of multiple antigens in a single electrophoretic location. The ratios of intensities from the kringle IV repeats to non-kringle IV components of apo(a) will facilitate a more accurate measurement of Lp(a) subform size, when compared to a known kringle IV/non-kringle IV standard.
  • the signal-producing molecules may include fluorescent tags. Fluorescence tagging and the detection of natural fluorescence in molecules is a method of analytical chemistry and biology that is well known in the art.
  • the instruments used to detect fluorescence may include the following components.
  • a light source with a broad optical bandwidth such as a light bulb or a laser is used as the source of the stimulating light.
  • An optical filter is used to select the light at the desired stimulation wavelength and beam it onto the sample.
  • Optical filters are available at essentially any wavelength and are typically constructed by the deposition of layers of thin film at a fraction of the wavelength of the desired transmission wavelength. The light that exits the optical filter is then applied to the sample to stimulate the fluorescent molecule.
  • the molecule then emits light at its characteristic fluorescent wavelength.
  • This light is collected by a suitable lens and is then passed through a second optical filter centered at the characteristic wavelength before being brought to a detection device such as a photomultiplier tube, a photoconductive cell, or a semiconductor optical detector. Therefore, only light at the desired characteristic wavelength is detected to determine the presence of the fluorescent molecule.
  • the at least two lipoprotein-binding complexes may include fluorescent molecules that emit light at different, distinguishable fluorescent wavelengths.
  • Fluorescent tags may be multiplexed in a single area such that they are optically distinct. For example, 5 different fluorescent tags, red, green, blue, yellow, and orange may be applied to the same limited area and be independently detected and distinguished by optical detection software.
  • the Life Technologies Alexa Fluor product line includes at least 19 distinct dyes that may be combined for tagging distinct antibodies to label and identify individual antigens. For example, as shown in the Examples described herein, Alexa 647, Alexa 546 and Alexa 488 may be combined for tagging distinct antibodies to label and identify individual antigens (e.g., Apo B, Apo C-III, and Apo E).
  • Additional fluorophores such as Alexa 430 may be included to optimize a method and avoid cross-talk between labels.
  • An optical system can quantitate the fluorescent signals and automatically normalize the signal value to generate relative densities or particle numbers. For example, by normalizing the extinction/emission coefficients or quantum relativity of each dye, relative values for concentration or particle number can be determined.
  • the system and methods may also include a device or use of a device for detecting the detectable signal, where the detecting indicates the level of the specific Lp(a) particles in the biological sample.
  • the device may also quantitate the level of specific Lp(a) particles based on the detection of the signal- producing molecule.
  • the presence of the detected particle or a portion thereof in the electrophoretic gel may then be quantified by measurement of the detectable signal or moiety.
  • the particle number may then be calculated according to known stoichiometric relationships.
  • the particle number may be quantified by comparison with a separate analysis that characterizes the total lipid particle or class of lipid particle concentration in the sample.
  • Such separate analysis may be ultracentrifugation, NMR, or any other analysis method that can characterize a concentration or total particle number for particles in the sample.
  • Said sample used in lipid particle electrophoresis and lipid particle quantification may be different aliquots of the same sample.
  • a suitable method for such in situ detection include a method of assessing the level of specific Lp(a) subform particles present in a biological sample. This method includes the steps of providing a biological sample containing Lp(a) subform particles and providing at least two lipoprotein-binding complexes. Each complex includes an antibody that binds a Lp(a) subform particle or a portion thereof, where the antibody is bound to a signal-producing molecule capable of producing or causing production of a detectable signal. Each detectable signal of the at least two lipoprotein-binding complexes is
  • This method also includes contacting the biological sample with the antibody under conditions suitable to form a lipoprotein-antibody-signal producing molecule complex and separating the lipoprotein particles present in the biological sample by depositing the biological sample on an electrophoretic gel and carrying out gel electrophoresis.
  • This method further includes detecting the detectable signal produced by the signal-producing molecule of the lipoprotein-antibody-signal producing molecule complex on the electrophoretic gel and determining the level of the specific Lp(a) subform particle present in the biological sample based on the detecting.
  • a further method for in situ detection includes a method of determining whether a subject is at increased risk for cardiovascular disease.
  • This method includes assessing the level of specific Lp(a) subform particles present in a biological sample.
  • the assessing includes the steps of providing a biological sample containing Lp(a) subform particles and providing at least two lipoprotein- binding complexes.
  • Each complex includes an antibody that binds an Lp(a) subform or a portion thereof, where the antibody is bound to a signal-producing molecule capable of producing or causing production of a detectable signal.
  • Each detectable signal of the at least two lipoprotein-binding complexes is
  • the assessing step also includes separating the lipid protein particles present in the biological sample by depositing the biological sample on an electrophoretic gel and carrying out gel
  • the method also includes the step of correlating the determined level of the Lp(a) subform particle to a control or reference value to determine if the subject is at an increased risk for cardiovascular disease.
  • Methods according to aspects described herein may also include measuring the particle number of the individual Lp(a) subforms; determining the size distribution of the Lp(a) subforms in the sample based on the determined molar mass and particle number of the individual Lp(a) subforms; and determining a cardiovascular risk value for the subject based on the determined size distribution of the Lp(a) subforms. Methods according to aspects described herein may also include measuring the particle number of the Lp(a) present in the sample and determining a cardiovascular risk value for the subject based on the measured particular number.
  • a method for predicting cardiovascular health involves obtaining a sample comprising Lp(a) subforms from a patient; separating the Lp(a) subforms in the test sample along an electrophoretic gel; measuring particle number of individual Lp(a) subforms in the sample; and determining a cardiovascular risk value for the subject based on the measured particle number of the Lp(a) subforms.
  • the cardiovascular risk may be determined as, for instance, low (or optimal), moderate, or high, as described above.
  • Methods described herein may also be carried out where the subject has, or is undergoing, an existing therapeutic regimen. Such methods may involve modifying the therapeutic regimen based on the determined size distribution of Lp(a) subforms, the particle number of the Lp(a), or both.
  • the therapy regimen may also be modified based on a determined cardiovascular risk value, as described above. For instance, the dosage of a statin (or other drug, as described herein) may be increased if the cardiovascular risk value is intermediate or high.
  • the invention also includes selecting a therapy regimen based on the risk for cardiovascular disease determined. For instance, an individual may be determined to be at an elevated risk according to the methods and a treatment regimen may then be selected based on the elevated risk.
  • the selected therapy regimen may include administering drugs or supplements.
  • Suitable drugs or supplements include those administered for the purpose of lowering serum cholesterol, lowering LDL, IDL, and VLDL, Lp(a) and/or raising HDL, as known in the art.
  • Suitable drugs include niacin, an anti-inflammatory agent, an antithrombotic agent, an anti-platelet agent, a fibrinolytic agent, a lipid reducing agent, a direct thrombin inhibitor, a glycoprotein lib/Ilia receptor inhibitor, an agent that binds to cellular adhesion molecules and inhibits the ability of white blood cells to attach to such molecules, a calcium channel blocker, a beta-adrenergic receptor blocker, an angiotensin system inhibitor, and
  • the selected therapy regimen comprises administering a drug selected from the group consisting of niacin, fenofibrate, estrogen, and raloxifene.
  • the selected therapy regimen includes niacin.
  • the selected therapy regimen includes a statin.
  • the selected therapy regimen includes administering niacin and a statin.
  • the selected therapy regimen includes administering a statin and ezetimibe.
  • the selected therapy regimen includes administering niacin, ezetimibe, a statin, or a combination thereof.
  • the selected therapy regimen may also involve giving
  • a report may also be generated that includes, among other things, a description of the selected treatment regimen.
  • the results of lipoprotein analyses are reported in such a report.
  • a report refers in the context of lipoprotein and other lipid analyses to a report provided, for example to a patient, a clinician, other health care provider, epidemiologist, and the like, which includes the results of analysis of a biological specimen, for example a plasma specimen, from an individual.
  • Reports can be presented in printed or electronic form, or in any form convenient for analysis, review and/or archiving of the data therein, as known in the art.
  • a report may include identifying information about the individual subject of the report, including without limitation name, address, gender, identification information (e.g., social security number, insurance numbers), and the like.
  • a report may include biochemical characterization of the lipids in the sample in addition to Lp(a), for example without limitation triglycerides, total cholesterol, LDL cholesterol, and/or HDL cholesterol, and the like.
  • a report may further include characterization of lipoproteins, and reference ranges therefore, conducted on samples prepared by the methods provided herein.
  • reference range refers to concentrations of components of biological samples known in the art to reflect typical normal observed ranges in a population of individuals.
  • Exemplary characterization of lipoproteins in an analysis report may include the concentration and reference range for VLDL, IDL, Lp(a), LDL and HDL, and subclasses thereof.
  • a report may further include lipoprotein size distribution trends.
  • the invention also may further include administering the selected treatment regimen to the subject. Accordingly, a further aspect of the present invention relates to a method of treating a subject having an elevated risk for cardiovascular disease determined according to methods described herein.
  • the invention also relates to a method of monitoring the risk for developing cardiovascular disease.
  • This method includes determining whether a subject is at increased risk for cardiovascular disease at a first time point and repeating the determining at one or more later time points (e.g., before and after therapeutic intervention or at progressive time points during a course of therapeutic intervention). The determined risk at each progressive time point is compared the determined risk from one or more earlier time points to evaluate whether the subject's risk for developing cardiovascular disease has increased or decreased, thereby monitoring the risk for developing cardiovascular disease.
  • This method may involve assigning a risk category based on the determined risk for developing cardiovascular disease and comparing the risk categories assigned at progressive time points (e.g., comparing a first risk category determined at a first time point to a second risk category taken at a second time point), thereby monitoring the risk for developing cardiovascular disease.
  • a method predicting cardiovascular health involves obtaining a sample from a patient; measuring the size distribution of Lp(a) subforms in the sample;
  • a therapeutic regimen is prescribed to the patient to reduce the risk of cardiovascular disease.
  • Blood samples were acquired from preferably fasting patients. At least 125 ⁇ , of serum was prepared from each of the blood samples using well-known methods in the art. Each sample was held at 2-8°C and tested within 4 days of collection.
  • the stain is comprised of Acid Violet Stain.
  • Preparation for Use Dissolve the dry stain in 1 liter of 10% acetic acid (lOOmL acetic acid into 900mL diHbO) and mix thoroughly. Fill the SPIFE stain vat.
  • Citric Acid Destain (Helena 551959)
  • the destain contains 0.3% (w/v) citric acid.
  • the powder contains Tris base with Tris-HCl and sodium Chloride. Dissolve the powder in 8L of deionized water and mix until dissolved.
  • Preparation for Use Use as is in preparation of Dilution Solution.
  • albumin dilution solution was used to dilute samples beyond the apoB linearity of the Lipo-IFE assay. Diluting with this solution maintains the surface tension relationship necessary for appropriate sample deposition.
  • Quality control samples were prepared from lyophilized serum reconstituted in 1.5 ml of deionized water, swirling gently and incubating in a rocker for 15 minutes. 200 ⁇ aliquots were portioned into tubes. An abnormal and a normal control were run on each gel with QC solutions. For sample preparation, in aliquot tubes, the first two positions were reserved for QC samples. The sample tray was loaded into an automated plate carrier (Hamilton). The samples were loaded into the SPIFE 3000 sample tray.
  • Helena were prepared by removing protective guards and positioned on the instrument. The Applicator Blades were loaded onto the instrument and the reagent vial placed into position. 0.5-1 mL REP Prep (Helena) was dispensed onto the electrophoresis chamber floor prior to putting the SPIFE Vis Cholesterol Gels on to the chamber floor. The gel was positioned on alignment pins and electrodes were positioned on the pins to complete the circuit between gel and DC power supply. Using the initial blotter (Helena), the edge of the blotter was lined up with the edge of the Mylar backing and the blotter was aligned between the gel blocks. The blotter was removed from the gel from the side of the gel initially encountering the blotter.
  • the "Gel Block Remover” was used to scrape off the gel-blocks at the cathodic and anodic ends of the gel.
  • the Rigid Antisera Template Helena
  • 250 of working ApoB antisera was dispensed in each sample lane on the rigid template.
  • the "comb blotter” was placed on the anode port of the Rigid Antisera Template and excess antisera removal is observed; it is allowed to remain in position for at least 30 seconds. The comb blotter was removed and the Rigid Antisera Template is carefully removed from the gel.
  • a blotter was roll positioned on the gel, minimizing trapped air bubbles, and two more blotters are placed on top.
  • the Rigid Antisera Template was placed upon the chamber floor alignment pins and the gel was press-blot by center-placing a weight upon the rigid template for 60 seconds.
  • TBS Tris-Buffered Saline
  • the blotting method was repeated and the gel was placed into the shallow container-bath of TBS solution (approximately 50mL), making sure the agarose side was facing up.
  • the wash tray was manually agitated or placed on a shaker for 1 minute.
  • the gel was removed from the bath and excess TBS is removed from the gel by gently shaking.
  • the second blotting method was repeated again, and one final time after dispensing approximately 0.5-1 mL REP Prep onto the left side of the electrophoresis chamber.
  • fractions present in the gel lanes were evaluated with the Quick Scan 2000, performing neutral density densitometer scans as directed in the Quick Scan 2000 Operator Manual. Each scan consisted of 3 fractions, Lp(a)-P, vLDL-P, and LDL-P.
  • Helena densitometer software automatically calculates and prints the relative percent and the mg/dL for each band when the specimen total Apo-B is entered as (% of Fraction) x (total Apo-B).
  • Doublets are seen on the gel when there is sufficient kringle/MW difference between the apo(a) of the Lp(a)-Particles to match the resolving ability of the system.
  • This system is adequate to probe and validate clinical significance of Lp(a)-subforms.
  • the gels can be modified to increase resolution if necessary by methods known in the art. The system identifies both bands for particle number and size.
  • FIGS. 1 A and IB show variable Lp(a)-P migration rates via the results of a Lipo-IFE protocol, as described herein.
  • FIG. 1A provides a basic example gel for evaluating differences in Lp(a) migration.
  • FIG. 1A labels electrophoretic banding on the example gel corresponding to LDL, vLDL, median Lp(a), slower cathodic Lp(a), and faster anodic Lp(a).
  • FIG. IB the results of 5 samples run according to the protocols in this application are shown. Samples all contain LDL and VLDL, and all but the negative control further includes Lp(a) particles.
  • the reference (known) Lp(a) content comprises an apo(a) moiety of 600-650 kD.
  • an "anodal" sample in lane 2 contains Lp(a) with an apo(a) moiety of greater than 650 kD
  • the "mid” sample in lane 3 contains apo(a) moieties of the same mass as the reference, 600-650kD
  • the "cathodal” sample contains apo(a) moieties of less than about 600 kD.
  • FIGS. 1A and IB illustrate the principle of differential detection and quantification of Lp(a) subforms.
  • FIG. 2 shows examples of samples with differential Lp(a)-P migration bias on zonal gels, separated via the Lipo-IFE protocol described herein.
  • a series of samples have been run on a gel in parallel on the Lipo-IFE system.
  • the cathode and anode ends of the gel are labeled and solid lines represent the expected position of Lp(a) particles after separation.
  • Four samples have been highlighted (sample numbers 10, 73, 24, and 44).
  • samples 10 and 73 show distinct Lp(a) subform size difference due to the migration rates of smaller subforms (samples 10 and 73, with dashed outline and positioned toward the cathodal end of the substrate) and larger subforms (samples 24 and 44, with a solid outline and positioned toward the anodal end of the substrate).
  • FIG. 3 compares the zonal gel (Lipo-IFE protocol, inset) shown in FIG. 2 to the same samples in a western blot.
  • samples 24, 73, 44, and 10 are further analyzed in a western blot analysis after apo(a) removal from the Lp(a) particles.
  • Western Blot analysis was carried out using standard protocols with Apolipoprotein(a) Isoform Analysis (AAISO), using the Novex ® WesternBreezeTM Chromogenic Western Blot Immunodetection Kit (Invitrogen Life Technologies).
  • AAISO uses electrophoresis and western blot to measure Apo(a) isoforms in serum or EDTA plasma. Serum (or plasma) is first reduced in dithioerythritol and 6-aminocaproic acid then denatured in beta mercaptoethanol. The denatured sample is then loaded onto a 4% Tris Glycine gel and
  • Samples 44 and 10 repeat the pattern with an anodal Lp(a) similarly having larger apo(a) bands at -700 kD and a cathodal Lp(a) having a smaller apo(a) band at less than 450 kD on the western blot.
  • FIGS. 4A and 4B show more examples of the Lp(a)-P migration differentials in a high-throughput run.
  • zonal gels run in parallel show variation among patient samples in a high-throughput experiment.
  • Anodal particles of more than about 650 kD and cathodal particles of less than about 600 kD are identified.
  • Anodal samples include sample numbers 1 147, 1200, 0481, 1621, 2420, 2495, and 2618.
  • Cathodal samples include sample numbers 1 101 and 261 1.
  • FIG. 5 shows comparison of apo(a) content and apoB content in a series of samples on a zonal gel.
  • five samples were probed by anti- apoB sera and anti-apoA sera, showing doublet banding, a product of some subjects having two sizes of apo(a) moieties on their Lp(a) particles.
  • Lp(a) doublet banding is seen on samples 1 , 3, 4 & 5; these samples contain both apoB & apo(a) probe response without non-specific protein residue in saline lane. Doublet banding is not artifactual.
  • Lp(a) doublet banding can be equal, primarily cathodal, or anodal with differential resolution.
  • Sample 2 has single Lp(a), but contains both apoB and apo(a) response, cathodal to LDL and without nonspecific protein residue in the saline lane.
  • the frequency of doublet banding ⁇ 1%.
  • Saline probe is used to establish presence of artifactual banding.
  • FIGS. 6A-6R present data comparing Lp(a)-P zonal migration velocities (inset, from FIGS. 2, 4A, and 4B) associated with increasing MW of apo(a), measured by western blot.
  • FIGS. 6A-6R more than 100 samples with Lp(a) zonal migration biases were compared to apo(a) isoform size analysis by western blot analysis, as described above.
  • the results from 19 experiments reflecting the same setup and analysis as shown in FIG. 3 are presented. The results show consistent agreement of the new zonal gel method for analyzing Lp(a)-P subform size with the more intensive analysis of separated apo(a) moieties from the same particles.
  • Example 3 Validation of Lp(a) Subform Size Identification Using Zonal Gel
  • Lp(a) subform size identification using zonal gel immuno-fixation electrophoresis was validated using an assay employing Meridian Custom conjugated Anti-apoB 100*-Alexa488 antibodies.
  • Custom conjugated antibodies were prepared and examined to show the distinction between apoB-containing particles.
  • HDL production Calbiochem Anti-apoB was purified and conjugated to Alexa488 fluorophore.
  • Three 500uL pilot lots were prepared to study signal:noise ratios. Lots were prepared where lot Al contained a 5: 1 dye to polyclonal antibody ratio, lot A2 contained a 7.5: 1 ratio, and lot A3 contained a 10: 1 ratio. All Concentrations were normalized to antibody titer for existing working Calbiochem antisera. Serums were chosen with variable Lp(a), VLDL, and LDL concentrations.
  • FIG. 7 shows fluorescent imaging for labeled anti-apoB antibodies bound to Lp(a), LDL, and VLDL particles.
  • the image is a 3Blot/TBS-Image (wet) (the dried gel was opaque and unsuitable for imaging).
  • the top row shows sample 3022 at the dilution levels of 5: 1 (Al), 7.5: 1 (A2), and 10: 1 (A3) Alexa Fluor® fluorescent dye:polyclonal antibody, in respective columns.
  • the same experiment is shown for sample 3052 in the bottom row. There are no observable differences in optical properties between the dilution ratios.
  • FIGS. 8A-8E show results of an initial comparison of acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL-P in a single sample.
  • FIG. 8A shows a native zonal gel separation of sample numbers 3022 and 3052, in respective columns containing duplicate runs, labeled by acid violet staining. There are 4 total sample runs, where the LDL is evident as a strong band and Lp(a) as a weak band below it.
  • FIG. 8B shows optical imaging with fluorescence of the same samples, where 3022 is on the top row and 3052 is on the bottom row.
  • FIG. 8C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample.
  • FIG. 8D is the optical density reading of the acid violet staining of sample 3022 in profile and
  • FIG. 8E is the optical density reading of the fluorescent conjugate of sample 3022 in profile (in the reverse direction of the A/V profile).
  • FIGS. 9A-9E show results of an initial comparison of acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 9A shows a native zonal gel separation of samples 3034 and 3051, in respective columns containing duplicate runs, labeled by acid violet staining.
  • FIG. 9B shows optical imaging with fluorescence of the same samples, where 3034 is on the top row and 3051 is on the bottom row.
  • the columns correspond to different fluorescent dye:anti-apoB ratios in reagents
  • FIG. 9C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low- density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 9D is the optical density reading of the acid violet staining of sample 3051 in profile
  • FIG. 9E is the optical density reading of the fluorescent conjugate of sample 3051 in profile (in the reverse direction of the A/V profile).
  • FIGS. 10A- 10E show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample. Both LDL, VLDL and Lp(a) were probed with anti-apoB-488*. The probe on the right of the "A" figures was an anti-apo(a) with A/V staining; this probe confirms the identity of the band as an Lp(a)-P.
  • FIG. 10A shows a native zonal gel separation of sample 0816, in the left column, labeled by acid violet staining. On the left, anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and
  • FIG. 10B shows optical imaging with fluorescence of the same sample where labeled. The columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al , A2, and A3, previously described.
  • FIG. IOC presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low-density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIGS. 1 lA-1 IE show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample. The distinction in data is the same as for FIG 10.
  • FIG. 1 1A shows a native zonal gel separation of sample 2377, in the left column, labeled by acid violet staining.
  • anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and VLDL.
  • anti-apo(a) antibodies are used, which only label the Lp(a) particles.
  • FIG. 1 I B shows optical imaging with fluorescence of the same sample where labeled. The columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al, A2, and A3, previously described.
  • FIG. 11C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low-density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 1 ID is the optical density reading of the acid violet staining of sample 2377 in profile
  • FIG. 1 1 E is the optical density reading of the fluorescent conjugate of sample 2377 in profile (shown in reverse sequence from A/V).
  • FIG. 12A-12E show results from a sample comparing the distinction in anodal (large) Lp(a) subforms and cathodal (small) Lp(a) subforms with acid violet staining detection methods (A/V) and apoB* fluorescently-tagged antibody labeling methods, for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • A/V acid violet staining detection methods
  • apoB* fluorescently-tagged antibody labeling methods for Lp(a)-P distinction from LDL and VLDL in a single sample.
  • FIG. 12A shows a native zonal gel separation of sample 3389, in the left column, labeled by acid violet staining.
  • anti-apoB antibodies are used for labeling, which are found on all of Lp(a), LDL, and VLDL.
  • FIG. 12B shows optical imaging with fluorescence of the same sample where labeled. The columns correspond to different fluorescent dye:anti-apoB ratios in reagents Al, A2, and A3, previously described.
  • FIG. 12C presents numerical results of the optical density readings of each sample, where %Lpa means percent Lp(a) (lipoprotein a) of total detected sample and %LDL means percent LDL (low-density lipoprotein) of the total detected sample. Further detailed analysis show the levels of Lp(a), VLDL, and LDL below the Lp(a)/LDL levels.
  • FIG. 12D is the optical density reading of the acid violet staining of sample 3309 in profile
  • FIG. 12E is the optical density reading of the fluorescent conjugate of sample 3389 in profile (shown in reverse sequence from A/V).
  • FIGS. 13A-13C shows a summary of figures 10- 12, comparing doublet-containing samples 0816, 3389, and 2377 to each other on adjacent gels.
  • FIG. 13 A shows the zonal gels labeled with acid violet stain for each sample, with apoB labeled in the first column and apo(a) labeled in the second column.
  • FIG. 13B shows the fluorescence detection for each sample, with the samples clearly labeled.
  • FIG. 13C presents the table summarizing each sample and its relative proportion of anodal (large) Lp(a) subform and cathodal (small) Lp(a) subform along with the LDL and VLDL portions of the sample. The methods show good agreement in calculated ratios of each lipoprotein type level.

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Abstract

Selon un aspect, la présente invention porte sur un procédé de détermination de la composition de sous-formes Lp(a) individuelles dans un échantillon d'analyse. Le procédé entraîne l'utilisation d'un échantillon d'analyse comportant des sous-formes Lp(a) prélevé sur un sujet ; la séparation des sous-formes Lp(a) de l'échantillon d'analyse le long d'un gel électrophorétique ; la mesure de la vitesse de migration des sous-formes Lp(a) individuelles le long du gel électrophorétique ; la comparaison, sur la base de ladite mesure, de la vitesse de migration des sous-formes Lp(a) individuelles avec une valeur de référence ; la détermination, sur la base de ladite comparaison, de la masse molaire des sous-formes Lp(a) individuelles. La présente invention porte également sur des procédés de prédiction de santé cardio-vasculaire.
EP14828097.7A 2013-12-18 2014-12-18 Identification de dimension de sous-forme lp(a) à l'aide d'électrophorèse d'immuno-fixation de gel zonal Withdrawn EP3084447A1 (fr)

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